Virulence and antifungal susceptibility of microsatellite genotypes of Candida albicans from superficial and deep locations

A set of 185 strains of Candida albicans from patients with vulvovaginal candidiasis (VVC) and from non-VVC clinical sources in southwest China was analysed. Strains were subjected to genotyping using CAI microsatellite typing and amplification of an intron-containing region of the 25S rRNA gene. Microsatellite genotypes of strains from non-VVC sources showed high polymorphism, whereas those of VVC were dominated by few, closely similar genotypes. However, among non-VVC strains, two genotypes were particularly prevalent in patients with lung cancer. 25S rDNA genotype A was dominant in VVC sources (86.7%), whereas genotypes A, B, and C were rather evenly distributed among non-VVC sources; known genotypes D and E were not found. In an experimental mouse model, isolates from lung cancer and AIDS patients proved to have higher virulence than VVC strains. Among 156 mice infected with C. albicans, 19 developed non-invasive urothelial carcinoma. No correlation could be established between parameters of virulence, source of infection, and incidence of carcinoma. C. albicans strains from VVC were less susceptible to itraconazole than the strains from non-VVC sources, whereas there was small difference in antifungal susceptibility between different 25S rDNA genotypes of C. albicans tested against amphotericin B, itraconazole, fluconazole, and flucytosine.     

The effect of histatin 5, adsorbed on PMMA and hydroxyapatite,on Candida albicans colonization

The limited number of treatments for oral candidiasis resulted in the emergence of azole‐resistant Candida albicans strains, thus enforcing the need for novel antifungal treatments. Although histatin 5 (H5) demonstrates antifungal activity, its inhibitory effect when adhered to hydroxyapatite and Polymetylmethacrylate (PMMA) surfaces, resembling conditions of the in vivo pellicle, remains unexplored. The objective of this in vitro study was to determine whether surface‐adhered H5 inhibits the colonization of C. albicans on hydroxyapatite and/or PMMA. The C. albicans assay involved developing a mono‐protein pellicle (either H5 or albumin) on hydroxyapatite and PMMA discs, introducing C. albicans and counting the number of adhered cells, throughout time, using scanning electron microscopy. A negative binomial statistical model and the Tukey–Kramer test were used for statistical analysis, with p < 0.01 indicating significance. H5‐coated PMMA had significantly reduced number of cells compared to albumin‐coated PMMA at 30, 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly in 90 and 1440 min (p < 0.0001). Similarly, H5‐coated hydroxyapatite had significantly fewer cells compared to the albumin‐coated surface at 90 and 1440 min (p < 0.0001), with the number of cells decreasing significantly at 30, 90 and 1440 min (p < 0.0001). In conclusion, C. albicans colonization was most inhibited by PMMA and hydroxyapatite‐adhered H5 after 1440 min, illustrating the time‐dependent effect of H5. In addition, yeast cells colonized albumin‐coated PMMA, while dense hyphal networks formed on albumin‐coated hydroxyapatite, suggesting that C. albicans morphology is influenced by the surface available for albumin adhesion. Copyright © 2012 John Wiley & Sons, Ltd.     

Transformation of Candida albicans with a synthetic hygromycin B resistance gene

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2‐containing plasmids or single‐copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT‐1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild‐type C. albicans. We used PCR to fuse CaHygB or SAT‐1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild‐type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.     

The quorum‐sensing molecule E,E‐farnesol—its variable secretion and its impact on the growth and metabolism of Candida species

Diseases caused by Candida species are an increasing problem. Candida species are associated with high overall mortality, due to a variety of virulence factors such as the yeast‐to‐hyphal switch and proteolytic enzymes. The phenomenon of microbial communication known as quorum sensing also seems to play an important role. The main characteristics of the quorum‐sensing molecule E,E‐farnesol are well known for C. albicans. The present study focused on two questions. One of them concerned the secretion of E,E‐farnesol by C. albicans and involved a close examination of the effect of the medium (serum) and the origin of the isolates used. The second one dealt with the activity of E,E‐farnesol in non‐C. albicans species, such as C. tropicalis and C. parapsilosis, e.g. its impact on biofilm formation and growth. Under serum conditions, C. albicans produced up to 58% more E,E‐farnesol at 37 °C than at 30 °C. The growth of all isolates was reduced and delayed by the administration of E,E‐farnesol. Of all Candida species, C. tropicalis isolates were most strongly affected by the addition of E,E‐farnesol. Biofilm formation on polystyrene was affected by E,E‐farnesol treatment in all non‐C. albicans species and C. albicans. E,E‐farnesol exerts its main effect by altering the metabolic activity and growth inhibition of treated Candida species. The results obtained indicate that the presence of E,E‐farnesol in the environment not only regulates the morphology of the Candida species but also affects its fitness. In this regard, the secretion of E,E‐farnesol might provide an advantage for members of the microbial community. Copyright © 2010 John Wiley & Sons, Ltd.     

Diverse Profiles of N‐acyl Homoserine l‐Lactones,Biofilm, Virulence Genes and Integrons in Food‐Borne Aeromonas Isolates

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N‐acyl‐homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin‐layer chromatography (TLC) analysis and microtiter‐plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N‐butanoyl homoserine lactone (C4‐HSL) and N‐hexanoyl homoserine lactone (C6‐HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N‐pentanoyl homoserine lactone (C5‐HSL), N‐heptanoyl homoserine lactone (C7‐HSL), and N‐octanoyl homoserine lactone (C8‐HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7‐HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat‐labile cytotonic enterotoxin (alt), heat‐stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India.     

Biochemical characteristics,virulence traits and antifungal resistance of two major yeast species isolated from kefir: Kluyveromyces marxianus and Saccharomyces unisporus

Kefir yeasts were investigated for their possible virulence using an in vitro system. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of 46 biochemical traits showed that all kefir isolates formed clusters distinct from Candida albicans. No isolate showed N‐acetylglucosaminidase activity, which is involved in resistance against host immune systems. All Kluyveromyces marxianus isolates formed pseudohyphae and grew at 42 °C, whereas Saccharomyces unisporus isolates did not. None of the kefir yeasts had proteolytic or haemolytic activities. All K. marxianus isolates were susceptible to fluconazole, whereas S. unisporus isolates were classified as resistant.     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Antibiotic Resistance Traits and Molecular Subtyping of Staphylococcus aureus Isolated from Raw Sheep Milk Cheese

The main objective of the present research was to evaluate the antibiotic resistance profiles of Staphylococcus aureus isolated from raw sheep milk cheese. A total of 150 strains were isolated from curd cheese samples and identified as S. aureus. The survey on antibiotic resistance was carried out on 47 strains, selected among isolates showing differences in the banding pattern after Pulsed Field Gel Electrophoresis (PFGE) screening or, belonging at the same pulsotype but isolated from different cheese samples. On selected strains antimicrobial resistance against ampicillin, penicillin, cloxacillin, tetracycline, erythromycin, and vancomycin was assessed by broth microdilution method. The presence of the genes coding for antibiotic resistance and virulence factors (agr alleles, sea‐see, and tst) was also investigated by PCR. Thirty‐one isolates belonging to agrI and agrIII groups carried at least one gene coding for enterotoxins or toxic shock syndrome toxin. Approximately 60% of the selected strains were susceptible to the tested antibiotics. Twelve of 47 isolates showed multiple resistance against ampicillin and penicillin. Only 1 strain, represented by a unique PFGE profile showed simultaneous resistance to ampicillin, penicillin and cloxacillin. Single resistance against tetracycline was found in 5 isolates belonging to 2 different pulsotypes. The results of this study suggest that the recovery of S. aureus resistant strains in raw milk cheese samples is quite common but it is limited to few antibiotic classes, mainly β‐lactams and tetracyclines. None of the strains showed resistance to erythromycin and vancomycin.     

Selection of Dominant NSLAB from a Mature Traditional Cheese According to their Technological Properties and in vitro Intestinal Challenges

Abstract: Isolates (47) of lactobacilli from 5 different productions of Melichloro cheese were examined for potential use as adjunct cultures. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of whole‐cell proteins classified 29 isolates as L. paraplantarum and 18 as L. paracasei subsp. paracasei. Randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) analysis differentiated the L. paraplantarum and L. paracasei subsp. paracasei isolates at strain level and both, RAPD analysis and whole‐cell protein profiling provided useful information about the diversity of nonstarter lactic acid bacteria (NSLAB) in the different cheese productions. The isolates were slow acidifiers and about 70% of them degraded, preferentially α_s‐casein. The amounts of amino acids accumulated in the milk increased with the incubation time. A similar enzyme profile was exhibited by strains of both species, except for α‐mannosidase and α‐fucosidase, which were not detected in the L. paracasei subsp. paracasei strains. All strains grew in the presence of bile at 0.3% and the majority was able to withstand pH 2.5 and pancreatin at 0.1%. Moreover, all strains reduced cholesterol in vitro, with higher removal ability recorded for strains of L. paraplantarum. A narrow spectrum of antibacterial activity was recorded for 88% of the strains. Selected isolates with appropriate technological and interesting in vitro intestinal challenges could be used as adjuncts and deserve further studies. Practical Application: Strains selected by this study could be used as adjuncts to make the Melichloro cheese. Their contribution to cheese flavor is then going to be studied to select the most appropriate. Of course these strains have to be also studied for their probiotic potential, to say that we have a probiotic food.     

Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models

Listeria monocytogenes is an important foodborne bacterial pathogen that can colonize food processing equipment. One group of genetically similar L. monocytogenes strains (RAPD type 9) was recently shown to reside in several independent fish processing plants. Persistent strains are likely to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing the persistent RAPD type 9 invaded Caco-2 cells in lower numbers (10(2)-10(3) CFU/ml) as compared to the four other strains (10(4)-10(6) CFU/ml), including food and human clinical strains. In the D. melanogaster model, the two RAPD type 9 strains were among the slowest to kill. Similarly, the time to reach 50% killed C. elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level (approximately 10(2) CFU/g) than the Scott A strain (approximately 10(5) CFU/g) (P<0.05). The addition of NaCl has been shown to cause autoaggregation and increases adhesion of L. monocytogenes to plastic. However, growth in the presence of NaCl did not alter the behaviour of the tested L. monocytogenes strains in the virulence models. Overall, the two strains representing a very common fish processing plant persistent group (RAPD type 9) appear to have a lower virulence potential in all four virulence models than Scott A and a strain isolated from a clinical case of listeriosis.     

Biodiversity of Yeast Mycobiota in “Sucuk,” a Traditional Turkish Fermented Dry Sausage: Phenotypic and Genotypic Identification,Functional and Technological Properties

In this study, yeasts from Turkish fermented sucuks were identified and their functional and technological properties were evaluated. Two hundred fifty‐five yeast isolates were obtained from 35 different sucuk samples from different regions of Turkey. The yeast isolates were determined as genotypic using 2 different polymerase chain reaction (PCR) methods (rep‐PCR and RAPD‐PCR). Functional and technological properties of including proteolytic, lipolytic, and catalase activities, tolerance to NaCl and bile, as well as growing rates at different temperature and pH conditions selected yeast strains were also evaluated. Candida zeylanoides and Debaryomyces hansenii were dominant strains in sucuk samples. All C. zeylanoides and D. hansenii tested could grow at the condition of 15% NaCl and 0.3% bile salt. However, none of the strains were able to grow at 37 °C, even though catalase activity, weak proteolytic and lipolytic activities was still observed. D. hansenii were able to grow only at pH 3, while some of C. zeylanoides could grow at lower pH levels (pH 2). Three and 4 strains of C. zeylanoides showed β‐hemolysis activity and nitrate reduction ability to nitrite, respectively. D. hansenii did not have properties, which are β‐hemolysis, nitrate reduction, or hydrogen sulfide production. Overall, diverse yeast mycobiota present in Turkish fermented sucuk and their functional and technological properties were revealed with this study.     

Persistence of Listeria monocytogenes strains isolated from products in a Polish fish-processing plant over a 1-year period

Seventy-one presumptive Listeria monocytogenes strains were isolated over a year from 152 samples comprising raw fish (salmon, seatrout) and their products (mainly, vacuum-packed cold-smoked sliced salmon) in a selected Polish fish-processing plant. Contamination of raw materials was at the level of 4.3–15.4%, whereas final products revealed significantly higher contamination (up to 77.8%) than regarded by other studies as typical (up to 40%). Strains were identified using conventional microbiological methods (including API®LISTERIA tests) and the PCR technique (aimed at iap gene fragment detection). A random amplification polymorphic DNA (RAPD) technique was applied to analyse their intraspecies diversity. RAPD typing revealed an incidence of eight RAPD types. Three of them were isolated over 8–10 months during the plant monitoring. It suggested that they were a persistent element of ‘in-house’ microflora and the applied typing technique produced evidence that fish products could be probably contaminated at the last stages of fish processing (e.g. smoking, slicing, and/or packaging). Their occurrence was probably supported by clone selection caused by ineffective application of cleaning and sanitizing procedures. The possibility of colonization of the production environment by fish-originated L. monocytogenes was also proven. Strains that belonged to a dominant RAPD type were additionally subjected to restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). RFLP-PFGE confirmed intraspecies similarity of strains belonging to a dominant RAPD type. A subset of strains from salmon samples was also characterized by serotyping. Contrary to earlier reports, they belonged mainly (91.7%) to the serotype 4.     

STUDIES ON OCCURRENCE AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS FROM SELECT MEATS

A study was undertaken to assess the prevalence of Clostridium perfringens in meat and to characterize the isolates obtained in the study for virulence factors. A total of 211 meat samples of different animals (70 each of buffalo and goat and 71 of poultry) were screened and the highest occurrence of C. perfringens was observed in goat (91.4%) followed by poultry (70.4%) and buffalo (65.7%). Among the 116 isolates (buffalo‐32, goat‐37 and poultry‐45) of C. perfringens screened for the presence of enterotoxin gene by PCR, 9.3, 32.4 and 15.5% isolates of buffalo, goat and poultry, respectively, were found to possess enterotoxin gene. Screening of 15 enterotoxin gene possessing isolates for verocytotoxicity revealed that 12 isolates exhibited cytopathic effect while 3 isolates did not show any cytopathic effect in spite of the presence of enterotoxin gene. A total of 115 C. perfringens isolates were screened for other virulence markers, i.e., lecithinase and hemolysin. The results revealed that the majority of the isolates expressed these activities. Antibiogram studies of C. perfringens isolates using 16 antibiotics displayed multidrug resistance. The isolates showed resistance to streptomycin, ceftazidime, colistin sulfate, cephalothin, ampicillin and gentamicin. Whereas 100% sensitivity to ciprofloxacin, ofloxacin and nitrofurantoin was seen, moderate sensitivity was observed with tetracycline and sulfatriad.     

High prevalence of antimicrobial‐resistant Escherichia coli from animals at slaughter: a food safety risk

BACKGROUND: There has been concern about the increase of antimicrobial resistant bacteria and protection of animal and public health, along with food safety. In the present study, we evaluate the incidence of antimicrobial resistance among 192 strains of Escherichia coli isolated from faecal samples of healthy food‐producing animals at slaughter in Portugal. RESULTS: Ninety‐seven % of the pig isolates, 74% from sheep and 55% from cattle were resistant to one or more antimicrobial agents, with the resistances to ampicillin, streptomycin, tetracycline and trimethoprim–sulfamethoxazole the most common phenotype detected. Genes encoding resistance to antimicrobial agents were detected in most of the resistant isolates. Ninety‐three % of the resistant isolates were included in the A or B1 phylogenetic groups, and the virulence gene fimA (alone or in association with papC or aer genes) was detected in 137 of the resistant isolates. Five isolates from pigs belonging to phylogroup B2 and D were resistant to five different antimicrobial agents. CONCLUSION: Our data shows a high percentage of antibiotic resistance in E. coli isolates from food animals, and raises important questions in the potential impact of antibiotic use in animals and the possible transmission of resistant bacteria to humans through the food chain. © 2012 Society of Chemical Industry     

食品中蜡样芽孢杆菌的分离及携带毒力基因的检测

为了确定成都市食品中蜡样芽孢杆菌所携带的毒力基因情况,本实验对市售食品共采样130份,通过菌落在MYP培养基上形态特征对蜡样芽孢杆菌进行初步分离,再利用16Sr RNA测序结果进行比对分析,并检测分离菌株携带的管家基因gyr B、rpo B、Vrr A、gro EL进一步确认,最后检测分离菌株携带毒力基因情况。结果表明130份样品中共检出23株蜡样芽孢杆菌,检出率为17.7%;23株分离菌株中,蜡样芽孢杆菌的4个管家基因gyr B、rpo B、Vrr A、gro EL检出率为100%,毒力基因的检测结果表明,nhe B和ent FM在16株分离菌株中检出,检出率为69.6%;nhe A和nhe C在14株分离菌株中检出,检出率为60.9%;hbl D在11株分离菌株中检出,检出率为47.8%;cyt K在10株分离菌株中检出,检出率为43.5%;bce T在9株分离菌株中检出,检出率为39.1%;hbl A和hbl C在8株分离菌株中检出,检出率为34.8%;cer和ces在2株分离菌株中检出,检出率为8.7%;未发现分离菌株携带hbl B和Hly基因。研究结果表明市售食品中分离的蜡样芽孢杆菌毒力基因携带率较高,对食品安全具有潜在威胁,应当引起有关部门注意。     

Dissemination of Enterococcus faecalis and Enterococcus faecium in a Ricotta Processing Plant and Evaluation of Pathogenic and Antibiotic Resistance Profiles

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)‐polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed β‐hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic‐resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other.     

RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing

Fifteen wild yeast strains were isolated in two factories of a lager brewing company in Poland. Their identification with API 32C system showed mainly the presence of Candida sake species (7/15). To differentiate the isolates, randomly amplified polymorphic DNA (RAPD) with (GTG)(5), (GAC)(5), (GACA)(4) microsatellite primers and M13 core sequence (5'-GAG GGT GGC GGT TCT-3') were chosen. The results of patterns similarity are presented as dendrograms for each RAPD analysis and for overall patterns. On the overall patterns, all isolates identified as C. sake, except Strain No. 1, were regrouped in one cluster. Collection strain C. sake CBS 617 was similar in 46% to the cluster with six isolates (Strain Nos. 3, 6, 8, 11, 13, 14). The second reference strain C. sake CBS 159 and the Strain No. 1 were regrouped with other Candida species (collection strains) showing, respectively, only 20% and 42% of similarity to other C. sake strains. The similarity based on the overall dendrogram between isolate Nos. 3, 6, 8, 11, 13, 14 and C. sake CBS 617 was 49%. Between those strains and other Candida, the similarity was only 37%.     

Differentiation of lactococci from 2 Greek cheeses with protected designation of origin by phenotypic criteria and RAPD-PCR

Abstract: Seventy‐six lactococci isolates from 2 protected designation of origin (PDO) cheeses were studied for their acidification ability, proteolytic activity, and inhibitory activities as well as their intraspecies characterization by randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR). Fifty‐two of them were characterized as Lactococcus lactis subsp. lactis by the SDS‐PAGE of whole‐cell proteins. The test strains increased the amount of acid in milk from 6 to 24 h as well as the quantities of amino acids on incubation for 4 d. The majority of the isolates degraded preferentially α_s‐casein. The isolates from Feta differed from those of Graviera Kritis in respect of β‐casein degradation. This fragment was either not degraded or underwent a small degradation by lactococci from Feta. A stronger intensity of acidification for the isolates from Feta and a higher casein breakdown ability for those from Graviera Kritis were also recorded. Lactococci from Feta and Graviera Kritis inhibited, preferentially, the growth of Escherichia coli O157:H7, and Yersinia enterocolitica, respectively. A high heterogeneity among the isolates according to RAPD‐PCR was determined, as well as grouping of the isolates according to their source of isolation. Selected isolates from each cheese could be used as starters to make either Feta or Graviera Kritis.     

Characterization of Osmotolerant Yeasts and Yeast‐Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast‐like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S‐ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD‐PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast‐like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.     

Selection of antifungal protein-producing molds from dry-cured meat products

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillus niger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicillium chrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37 kDa for AS51D and 9 kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.